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Loss of ALS-linked gene C9orf72 affects motor skills early in study

Decline in activity in the C9orf72 The gene linked to amyotrophic lateral sclerosis (ALS) affected the neuromuscular junction – where nerve and muscle cells connect – in a zebrafish model, establishing the model as a tool for research into the disease and the gene’s role in the symptoms of ALS.

Zebrafish in this C9orf72 The loss-of-function model showed “motor defects, muscle atrophy, loss of motor neurons, and mortality in early larval and adult stages,” the scientists reported. As such, they expect the model to be useful for testing drugs aimed at restoring communication between nerve cells and muscles.

The study, “Reduced C9orf72 function leads to defective synaptic vesicle release and neuromuscular dysfunction in zebrafishwas published in Communications Biology.

ALS is characterized by the shrinkage and death of motor neurons that control voluntary muscle movement. A faulty C9orf72 which results in an abnormal protein of the same name, is the most common genetic cause of the disease, occurring in approximately 50% of hereditary cases and up to 10% of sporadic cases.

Analysis of tissues and cells isolated from ALS patients indicates that this genetic defect leads to a loss of C9orf72 function, but the exact mechanism leading to disease remains poorly understood.

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Studies in ALS patients and animal models suggest that this loss of function impairs the synapses that connect the end of motor nerve cells and muscle cells at the neuromuscular junction (NMJ) – the place where nerves and muscle cells communicate.

“This synaptic dysfunction is observed in all people with the disease and occurs before the death of motor neurons,” said Zoé Butti, first author of the study and doctoral student at the National Institute for Scientific Research (INRS), in a statement. .

To further explore these mechanisms, Butti and a research team based at INRS-Centre Armand-Frappier Santé Biotechnologie in Quebec, Canada, examined the impact of reducing C9orf72 gene activity on the zebrafish neuromuscular system.

It should be noted that the zebrafish is a standard research model. They have a similar genetic structure to humans, are easy to care for, and their eggs develop outside the mother’s body, making it easier to study early development.

After establishing the ALS zebrafish model – which was about half normal C9orf72 gene activity — researchers have found that most embryos die a few weeks after fertilization. Compared to healthy zebrafish, ALS fish had a 2-5% survival rate after 15 days. Those who survived without physical defects showed a significant decrease in motor activity.

An examination of their neuromuscular junctions at the time low motor activity became evident (six days post fertilization, usually abbreviated by scientists as dpf) revealed a significant reduction in synaptic markers in ALS fish, which had not been observed at earlier times.

“While the synaptic structures of the NMJ develop correctly and are preserved in the early larval stages in [ALS fish]they start to degenerate from 6 dpf,” the team wrote.

Other measurements showed a decrease in the frequency and strength of electrical nerve signals in the muscle cell connection at the neuromuscular junction.

Accumulation and clumping of a protein called TDP-43 in nerve and muscle cells is a hallmark of ALS. Typically, TDP-43 is found in the nucleus, but the muscle cells in the fish ALS model had this protein mostly outside the nucleus, in the cell cytoplasm.

However, when the mutant fish were given the genetic instructions to make a healthy C9orf72 protein, most of these alterations were rescued, becoming similar to a healthy group of control fish, which provided “strong evidence that C9orf72 silence in zebrafish recapitulates key pathology [disease-causing] hallmark of ALS,” the scientists wrote.

“In about 97% of ALS patients, the TDP-43 protein is depleted from the nucleus and forms aggregates in the cytoplasm rather than in the nucleus, as it does in healthy people,” Kessen said. Patten, PhD, study leader. author and ALS specialist at the INRS centre. “We want to dig deeper into this relationship between the two proteins.”

ALS fish that survived to adulthood showed impaired swimming behavior and swam with weak movements, showing signs of paralysis before dying. These fish also showed alterations in their NMJ, smaller mature motor neurons in the spinal cord, and significant shrinkage (atrophy) of muscle fibers, “consistent with the pathology of motor neuron degeneration in ALS patients”. wrote the researchers.

Analysis of spinal cord motor neurons in adult ALS zebrafish revealed that TDP-43 was depleted in the cell nucleus, but aggregated in the cytoplasm.

To understand the underlying molecular mechanisms of C9orf72 deficiency, the researchers also looked at protein production in ALS and control fish in the early embryonic stages.

Of the 24 proteins that were differentially produced in ALS fish and control fish, several were proteins required for proper synapse function. The main protein was SV2a, which is also downregulated in C9orf72-related ALS patient neurons. Experiments confirmed that SV2a interacts with the C9orf72 protein.

The SV2a protein is known to be involved in the release of synaptic vesicles, which carry chemical messengers (neurotransmitters) in the NMJ from nerve cells to muscle cells. The analysis showed that this process was significantly reduced in ALS fish, suggesting that C9orf72 plays a key role in regulating the release of synaptic vesicles from nerve cells.

The researchers also found that production of the protein Rab3a, essential for transporting synaptic vesicles to nerve cell synapses, was lower in ALS fish.

“In conclusion, we generated a stable C9orf72-LOF model in zebrafish that recapitulates some major features of ALS and improves our understanding of ALS pathogenesis,” the researchers wrote. “Importantly, our findings demonstrate that loss of C9orf72 function impairs synaptic function at the NMJs and leads to motor deficits.”

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